Melanoma is the most deadly form of skin cancer. Melanomas are highly heterogeneous, containing many different cell subpopulations within a single tumor. The ability of melanoma subpopulations to initiate tumors has been studied in transplantation assays. However, the requirement of subpopulations for tumor maintenance has not been directly examined. The proposed research aims to identify melanoma cell subpopulations that are important in tumor maintenance and drug resistance. For these studies I will use a zebrafish melanoma model in which the most common human melanoma oncogene, BRAFV600E, is coupled with a p53 loss-of-function mutation. Melanoma cell subpopulations, as defined by the expression of cell surface and other markers, will first be identified. Using a rapi and versatile system for generating transgenic zebrafish melanomas, I will label subpopulations then determine the extent to which they can initiate tumor formation in transplantation experiments. Such assays have been performed on human melanomas, but their ability to define subpopulations of cells responsible for tumor maintenance is unclear. To systematically assess whether subpopulations capable of melanoma initiation are important for tumor maintenance I will use a targeted cell ablation approach. With this approach, subpopulations will be ablated in intact melanomas and the consequences on tumor growth and persistence measured. The ability of subpopulations to resist death caused by the BRAF inhibitor PLX4032 will be also determined. Subpopulations resistant to PLX4032 will be genomically profiled and these profiles compared to those of PLX4032-resistant human tumors. Ablation of these subpopulations will be performed in conjunction with PLX4032 treatment to determine if such a combination could lead to a durable response. Ultimately my goal is to identify melanoma subpopulations that should be targeted by chemotherapies for effective and lasting cancer treatment.